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Biomedical Research and Therapy 2016, 3(7): 723-732 ISSN 2198-4093 www.bmrat.org
723 The role of Ficus virens Ait and its novel bioactive compound
ORIGINAL RESEARCH
Extenuating the role of Ficus virens Ait and its novel bioactive compound on antioxidant defense system and oxidative damage in cigarette smoke exposed rats Danish Iqbal1,2, M. Salman Khan1*, Amir Khan3, Saheem Ahmad1
1Clinical Biochemistry and Natural Product Research Laboratory, Department of Biosciences, Integral University Lucknow-226026, India 2Department of Medical Laboratory Sciences, College of Applied medical Sciences, Majmaah University, Al-majma’ah-11952, Saudi Arabia 3Department of Maxillofacial Surgery (Biochemistry), College of Dentistry, Taif University, KSA
*Corresponding author: [email protected]
Received: 21 June 2016 / Accepted: 13 July 2016/ Published online: 26 July 2016 ©The Author(s) 2016. This article is published with open access by BioMedPress (BMP)
Abstract— Introduction: Production of free radicals is associated with cigarette smoke (CS) which in turn generates oxidative stress, could be responsible for alterations in the activities of enzymatic and non-enzymatic antioxidants that links with atherosclerosis. Methods: Therefore, the putative preventive effects of F. virens extract and its bioactive compound (F18), n-Octadecanyl-O-α-D-glucopyranosyl(6’→1’’)-O-α-D-glucopyranoside were investigated on overall enzyme and non-enzymatic defense system and in oxidative stress CS-exposed rats. Results: The enzymatic activities of hepatic and lung CAT, SOD, Gred and GST in CS exposed rats were significantly decreased, while Gpx activity in CS exposed rats was increased. Similarly, hepatic and lung GSH content was reduced when compared to value of normal control group. Simultaneous administration of FVBM extract (50 and 100 mg/rat) and F18 bioactive compound (1 mg/rat) significantly increases hepatic and lung CAT, SOD, Gred and GST activity as well GSH concentration coupled with decrease in Gpx level in CS-exposed stress rats. Moreover, our histological observations concludes the pulmonary congestion, thickening of interalveolar septa and foci of collapsed alveoli with subsequent dilation of the adjoining alveolar spaces as well as development of large irregular spaces in rats lung exposed to cigarette smoke. Similarly, the liver also showed morphological alterations with congestion in central vein, portal inflammation and necrosis in CS-exposed rats. These morphological changes reversed significantly after treatment with FVBM extract and F18 compound. Conclusion: Thus biochemical and histopathological studies suggested that, FVBM extract and F18 showed its protective nature against CS-exposed rats. Keywords: Cigarette smoke, Oxidative stress, Antioxidant defense system, Histopathology, Ficus virens
INTRODUCTION
Oxidative stress is one of the major symptoms
accompanying physiological functions and many
pathological conditions such as cancer, diabetes,
chronic obstructive pulmonary disease, cardiovascular
and neurodegenerative diseases and also in the aging
process itself (Aruoma, 1998; Santilli et al., 2015; Sen et
al., 2010). Cigarette smoking (CS) contributes a
considerable amount of free radicals, estimated as 1014
and 1015 free radicals/puff in the tar and gas phases
(Church and Pryor, 1985) and is the major risk factor
as well as leading cause cardiovascular disease (CVD)
(Messner and Bernhard, 2014; WHO, 2015).
Moreover, CS generate free radicals that could be
responsible for alterations in the activities of
enzymatic viz., superoxide dismutase (SOD), catalase
(CAT), glutathione peroxidase (Gpx), glutathione
reductase (Gred), glutathione-s-transferase (GST) and
non-enzymatic, namely GSH, antioxidants (Ramesh et
al., 2007; Ramesh et al., 2015; Thirumalai et al., 2011).
DOI 10.7603/s40730-016-0033-5
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724 The role of Ficus virens Ait and its novel bioactive compound
Various studies suggested that medicinal plants are
source for the prevention of numerous oxidative stress
related diseases (Akhter et al., 2013; Alvi et al., 2015;
Khan et al., 2013; Salvamani et al., 2014). Thus, natural
compounds with antioxidant properties could
contribute to the protection of cell and tissue against
deleterious effects caused by CS generated reactive
oxygen species (ROS). Ficus species exhibits strong
antioxidant and biological properties, known to
diffuse the toxic free radical and can be used as a
possible protective agent for treatment of oxidative
stress related disorders (Iqbal et al., 2014a; Sirisha et
al., 2010; T S et al., 2013). We have previously shown
that Ficus virens bark methanolic (FVBM) extract
contained large amount of antioxidant with significant
hypolipidemic property (Iqbal et al., 2014b; Iqbal et al.,
2015). The current investigation demonstrates the
protective role of FVBM extract and its principal
bioactive compound, n-Octadecanyl-O-α-D-
glucopyranosyl(6’→1’’)-O-α-D-glucopyranoside in
cigarette smoke-induced oxidative stress.
MATERIALS AND METHODS
Chemical Reagents
Bradford dye was purchased from Sigma Aldrich, In-
dia, potassium dichromate, hydrogen peroxide (H2O2),
glacial acetic acid was procured from Merck Pvt Ltd,
India; capston cigarette from Capston, India ltd. All
other chemicals were procured either from Himedia
Laboratories, Mumbai, India or of analytical grade.
Isolation of Bioactive compound
Bioactive compound; n-Octadecanyl-O-α-D-
glucopyranosyl (6→1″)-O-α-D-glucopyranoside (F18)
from FVBM extract was isolated by following the pro-
tocol (Iqbal et al., 2015).
Animals
Male Sprague-Dawley (SD) rats weighed around 100-
150 gm were procured from Indian Institute of
Toxicology Research Center, Lucknow. The proposed
study was approved by Institutional Animal Ethics
Committee (IAEC) (registration number:
IU/Biotech/project/CPCSEA/13/11). The rats were
housed 5 per cage for one week in the animal house
for acclimatization at a temperature of 21-22˚C with 12
hours light and dark cycle. The rats were given
standard diet and water ad libitum.
Dose preparation
Sequentially extracted FVBM extract, its bioactive frac-
tion (F18) and reference drug atorvastatin were dis-
solved in 10 % dimethyl sulfoxide (DMSO) at different
concentrations and were homogenized with saline.
The doses of the extracts were selected on the basis of
previously published reports (Iqbal et al., 2015; T S et
al., 2013).
Diet/exposure to cigarette smoke
FVBM extract, its bioactive fraction (F18) and
atorvastatin suspension was administered through
gastric intubation in two divided doses (morning and
evening) of 0.5 ml each/rat/day. Rats in smoking
control group received 0.5 ml of saline containing 10
% DMSO (vehicle) twice daily while rats in normal
control group received 0.5 ml of saline containing 10
% DMSO twice daily. The rats were divided randomly
and equally (5 rats in each group) in groups as
illustrated in Table 1.
Table 1. Protocol for the treatment of cigarette smoke-
induced oxidative stress in rats
Group Treatment
N-C Normal control
S-C Cigarette smoking control + vehicle
FVT-1 Smoke-exposed + plant extract (FVBM) (50
mg/rat/day)
FVT-2 Smoke-exposed + plant extract (FVBM) (100
mg/ rat/day)
CT Smoke-exposed + bioactive compound (F18) (1
mg/ rat/day)
AT Smoke-exposed + standard (Atorvastatin) (1
mg/ rat/day)
Rats were exposed to cigarette smoke in the morning
by keeping two rats in bottomless metallic container
(10 x 11 x 16 inch), having two holes of 3 and 1.5 cm
diameter, one on the either side. A burning cigarette
was introduced through one hole (3 cm) and the other
hole (1.5 cm) was used for ventilation. Animals were
exposed to CS for 30 minutes, daily for 4 weeks with
interval of 10 min between each 10 min exposure,
using 3 cigarettes/day/2 rats in each group (Anbarasi
et al., 2006).
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725 The role of Ficus virens Ait and its novel bioactive compound
Collection of different organs
Liver and lung were excised and kept in ice-cold sa-
line. A portion of liver and lung were immediately
fixed in 10 % neutral formalin for histopathological
studies.
Preparation of homogenate and post mitochondrial
supernatant
At the end of the experiment, liver and lung from the
rats were promptly excised and chilled in ice-cold sa-
line. After washing with saline, it was blotted and
weighed. One gram of wet tissues was cut into pieces
and homogenized with 9 ml of chilled 0.1 M sodium
phosphate buffer, pH 7.4 (containing 1.17 % KCl) in a
waring blender. The homogenate was centrifuged at
1,000 rpm for 10 min at 4˚C, aliquoted and stored at
−20˚C. The remaining portion of the liver and lung
homogenates was centrifuged at 12, 000 rpm for 20
min at 4˚C. The post mitochondrial supernatant (PMS)
thus obtained was also aliquoted and stored at –20˚C
for future use.
Activities of antioxidant enzymes
The enzymatic activity of catalase in PMS of liver and
lung was measured by adopting the procedure of
Sinha (1972). Enzymatic activity of SOD in PMS
fraction of liver and lung was determined by the
method as described by Kakkar et al. (1984) based on
the 50 % inhibition of the formation of nicotinamide
adenine dinucleotide (NADH)-phenazine methosulphate-
nitroblue tetrazolium formazan at 560 nm. Glutathione
peroxidase activity in liver and lung homogenate was
assayed by modifying of the previous protocols
(Hafeman et al., 1974; Mills, 1959). The enzymatic
activity of Gred in liver and lung was determined
according to the method of Carlberg and Mannervik
(Eriksson et al., 1975). Method of Habig et al. (1974)
was used to measure the GST activity in PMS fraction
of liver and lung.
Activity of non-enzymatic antioxidant
For the determination of GSH content in liver or lung
homogenate, the previous methods were followed
(Ellman, 1959; Sedlak and Lindsay, 1968).
Histopathological studies of liver and lung
For histopathological study, a portion of liver and
lung were used. For microscopic preparation of the
above tissues, method of Disbrey and Rach (1970) was
used. Two formalin fixed samples from each tissue
were embedded in paraffin and sectioned after block
preparation. The paraffin block was sectioned in 4~5
μm thickness by using a microtome to make a slide
and then it was dyed with hematoxylin-eosin (H&E)
and observed under a light microscope and
photographs were taken (JA, 2001) .
Protein estimation
The protein concentration of liver homogenate and
PMS was analyzed by the method of Bradford (1976)
using bovine serum albumin as standard. Aliquots of
liver homogenates and PMS were first precipitated
with 10 % TCA followed by centrifugation at 1500
rpm for 10 min. The protein pellets were dissolved in
0.5 N NaOH and used for protein determination.
Data analysis
For all assays, samples were analysed in triplicate and
the results were expressed as mean ± SD. The results
were evaluated using one-way analysis of variance
(ANOVA) and two tailed Students T-test. Statistical
significance were expressed as *p<0.05, **p<0.01 and
***p <0.001.
RESULTS
Regulatory effects of FVBM extract, F18 bioactive
compound and atorvastatin on antioxidant defense
system in smoke-exposed rats after 4 weeks of
treatment
Free radicals released by cigarette smoke (CS)
generate oxidative stress. This process could be
responsible for alterations in the activities of
enzymatic and non-enzymatic antioxidants and
development of atherosclerosis. Since, protection
against oxidative stress/ROS is provided by enzymatic
and non-enzymatic antioxidants, therefore, the status
of antioxidant enzymes, such as CAT, SOD, Gpx, GST
and Gred including GSH concentrations in liver and
lung of experimental smoke-exposed rats are
important.
Impact on the regulation of hepatic and lung cata-
lase, superoxide dismutase, glutathione peroxidase,
glutathione reductase, glutathione-s-transferase ac-
tivities and reduced glutathione content
The enzymatic activities of hepatic CAT, SOD, Gred
and GST in S-C rats were significantly decreased from
N-C values of 62.28, 7.65, 18.29 and 132.8. U/mg
protein to 32.45, 4.8, 11.28 and 68.12 U/mg protein,
Iqbal et al., 2016 Biomed Res Ther 2016, 3(7): 723-732
726 The role of Ficus virens Ait and its novel bioactive compound
respectively, while Gpx activity in S-C rats was
increased from N-C values of 44.2 to 57.28 U/mg
protein (Fig. 1). The restoration in above enzymatic
activities of hepatic CAT, SOD, Gred and GST in FVT-
2 treated rat was 24, 32, 48 and 36 % of respective
normal control values. The values of these enzymes in
C-T group, in comparison to S-C values, were
significantly increased by 35, 53, 60 and 43 %
respectively. In contrast the hepatic Gpx activity was
significantly decreased by 14, 18, 20 and 10 % in FVT-
1, FVT-2, C-T and A-T rats. As depicted in fig. 3, the
hepatic GSH content were reduced by 43 % when
compared to value of N-C group. Administration of
FVBM extract, F18 bioactive compound and
atorvastatin to CS-exposed rats resulted in a
significant increase in GSH content by 22, 32, 45 and
18 % respectively.
Figure 1. Impact of FVBM extract,F18 bioactive compound andatorvastatin on Liver CAT, SOD, Gpx, Gred and GST activities in
cigarette smoke-exposed rats after 4 weeks of treatment. One unit (U/ mg protein) of Gpx activity is defined as nmole oxidized glutathione
formed/min/mg homogenate protein. One unit (U/ mg protein) of Gred activity is defined as nmole NADPH oxidized/min/mg PMS protein. One
unit (U/ mg protein) of GST activity is defined as the nmole of 1-chloro 2,4-dinitrobenzene (CDNB) conjugate formed/min/mg PMS protein. One unit
(U/mg protein) of CAT activity is defined as the μmoles of H2O2 decomposed/min/mg protein. One unit (U/mg protein) of SOD activity is defined as
the amount of enzyme required to inhibit O.D. at 560 nm of chromogen production by 50 % in one minute. Values are mean SD from
homogenate/PMS fraction of liver of 5 rats in each group. Significantly different from N-C at ap<0.001, significantly different from S-C at ap<0.001, significantly
different from S-C at bp<0.01, significantly different from S-C at cp<0.05.
Figure 2. Impact of FVBM extract, F18 bioactive compound andatorvastatin on lung CAT, SOD, Gpx, Gred and GST activities in
cigarette smoke-exposed rats after 4 weeks of treatment. One unit (U/ mg protein) of Gpx activity is defined as nmole oxidized glutathione
formed/min/mg homogenate protein. One unit (U/ mg protein) of Gred activity is defined as nmole NADPH oxidized/min/mg PMS protein. One
unit (U/ mg protein) of GST activity is defined as the nmole of 1-chloro 2,4-dinitrobenzene (CDNB) conjugate formed/min/mg PMS protein. One unit
(U/mg protein) of CAT activity is defined as the μmoles of H2O2 decomposed/min/mg protein. One unit (U/mg protein) of SOD activity is defined as
the amount of enzyme required to inhibit O.D. at 560 nm of chromogen production by 50 % in one minute. Values are mean SD from homoge-
nate/PMS fraction of Lung of 5 rats in each group. Significantly different from N-C at ap<0.001. Significantly different from N-C at bp<0.01. Significantly different
from S-C at ap<0.001. Significantly different from S-C at bp<0.01. Significantly different from S-C at cp<0.05.
Iqbal et al., 2016 Biomed Res Ther 2016, 3(7): 723-732
727 The role of Ficus virens Ait and its novel bioactive compound
Moreover, similar pattern was observed in lung en-
zymatic and non-enzymatic antioxidant defense sys-
tem. As depicted in Fig. 2 the enzymatic activities of
lung CAT, SOD, Gred and GST in S-C rats were signif-
icantly decreased from N-C value of 32.5, 14.7, 24.3
and 127.7 U/mg protein to 21.86, 9.56, 11.78 and 102.47
U/mg protein (Fig. 2). Whereas, lung Gpx activity in S-
C rats was significantly increased from N-C values of
59.68 to 82.73 U/mg protein. The restoration in above
enzymatic activities of lung CAT, SOD, Gred and GST
in FVT-2 treated rat was 34, 40, 41 and 20 % of respec-
tive N-C values. The values of these enzymes in C-T
group, in comparison to N-C values were significantly
increased by 42, 50, 47 and 24 % respectively. In con-
trast the lung Gpx activity was significantly decreased
by 11, 17, 26 and 16 % in FVT-1, FVT-2, C-T and A-T
treated rats. Similarly, lung GSH content which was
significantly reduced by 33 % in S-C rats as signifi-
cantly increased by 20, 37, 46 and 28 % after adminis-
tration of FVBM extract, F18 bioactive compound and
atorvastatin (Fig. 3). Here it is interesting to mention
that atorvastatin also exhibited significant ameliora-
tion in all the enzymatic and non-enzymatic activities
but is considered to be less effective than higher dose
of FVBM extract and F18 bioactive compound.
In summary, hepatic and lung CAT, SOD, Gred, GST
enzymes and non-enzymatic GSH, which constitute a
mutually supportive team of defense against ROS, are
significantly ameliorated after feeding of FVBM
extract and F18 bioactive compound as well as
substantially quenches the free radicals, thus
positively normalizing the above enzyme levels close
to normal values.
Figure 3. Effect of FVBM extract,F18 bioactive compound andatorvastatin on liver and lung GSH content in cigarette smoke-
exposed rats after 4 weeks of treatment. Values are mean (nmole SH group/mg protein) SD from homogenate of liver or lung of 5 rats in
each group. Significantly different from N-C at ap<0.001. Significantly different from S-C at ap<0.001. Significantly different from S-C at bp<0.01. Significantly different from S-C at cp<0.05.
Histopathological studies of liver and lung
It is apparent from Fig. 4A that histopathology of the
liver of the control rats showed normal looking uni-
form hepatocytes with small vesicular nuclei and ab-
undant eosinophilic granular cytoplasm with distinct
cell boundaries. Architecture of liver is well main-
tained. Normal Interstitial cells with vascularity. CS-
exposed rats showed smaller hepatocytes with small
vesicular or pyknotic nuclei and reduced granularity
of cytoplasm. Cytoplasmic boundaries are relatively
indistinct. Architecture not well maintained. Intersti-
tial cells increased and vascularity is reduced (Fig.
4B). Administration of FVBM extract at lower dose
has shown smaller hepatocytes with smaller vesicular
nuclei and eosinophilic cytoplasm with indistinct cell
boundaries showing proliferation. Architecture not
well maintained but vascularity has relatively in-
creased (Fig. 4C). Moreover, liver section of FVT-2 rats
(higher dose) (Fig. 4D) showed proliferated normal
hepatocytes with normal vesicular nuclei and abun-
dant eosinophilic cytoplasm with distinct cell bounda-
ries. Architecture well maintained with normal inters-
titial cell and vascularity. Similarly, liver section of C-
T rats showed proliferative hepatocytes with normal
morphology as well as architecture. Interstitial cells
normal with normal vascularity, also no toxic effects
noted (Fig. 4E). Furthermore, liver section of A-T rats
showed normal hepatocytes with normal vesicular
nuclei and abundant eosinophilic cytoplasm alongwith
well maintained normal architecture and vascularity (Fig. 4F).
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728 The role of Ficus virens Ait and its novel bioactive compound
Figure 4. Histology of liver section. A. Microphotograph of liver section from normal control rats, B. Microphotograph of liver
section from cigarette smoke-exposed rats after 4 weeks of cigarette smoke exposure, C. Microphotograph of liver section from
cigarette smoke-exposed rats after 4 weeks of FVBM-50 treatment, D. Microphotograph of liver section from cigarette smoke-
exposed rats after 4 weeks of FVBM-100 treatment, E. Microphotograph of liver section from cigarette smoke-exposed rats after 4
weeks of F18 bioactive compound treatment, F. Microphotograph of liver section from cigarette smoke-exposed rats after 4 weeks of
atorvastatin treatment. Figures were captured at 100X.
Histopathology of lung in N-C group shows normal
lung stroma and characteristic spongy appearance of
the lung with normal looking numerous alveolar
spaces, had normal blood vessels and bronchi. The
bronchoalveolar unit parenchyma in the normal lung
was within the limits (Fig. 5A). The lung section of CS-
exposed rats showed the increased volume of stroma
with presence of few collections of stromal cells with
reduced alveolar spaces, reduced vascularity and con-
stricted bronchi (Fig. 5B). There was obliteration of
most alveoli and subsequently compensatory dilata-
tion and expansion of the contiguous ones with de-
struction of alveolar wall. On treatment with lower
dose of FVBM extract, section of lung shows reduced
stroma, increased alveolar spaces, vascularity with
patent blood vessels and dilated bronchi (Fig. 5C). On
the other hand, treatment with higher dose, section of
lung showed changes as described above but bronchi
are relatively constricted (Fig. 5D). In addition, in lung
section of C-T rat observed that stroma is further re-
duced with presence of large alveolar spaces. Blood
vessels were normal but smaller bronchi were further
constricted (Fig. 5E). Furthermore, lung slice of ator-
vastatin treated group showed the alveolar spaces
comparatively less than normal with corresponding
increase in stromal elements. Blood vessels and bron-
chi were normal in the presence of few constricted
smaller bronchi (Fig. 5F).
DISCUSSION
Cigarette smoking (CS) is the foremost cause of
morbidity and mortality worldwide (Alam et al., 2013;
Jha et al., 2008) and is considered to be the preventable
risk factor for CVD (Ambrose and Barua, 2004).
Oxidative stress caused by CS are responsible for
enhanced lipid peroxidation, protein oxidation, DNA
damage and endothelial damage that could results in
various diseases such as CVD, stroke,different types
of cancer and diabetes (Messner and Bernhard, 2014;
Mohod et al., 2014; Valavanidis et al., 2009; Willi et al.,
2007).
Cigarette smoke is a complex situation possessing an
array of free radicals and ROS (Pryor and Stone, 1993).
These, free radicals are highly reactive molecules
occurs in normal consequence of a variety of
biochemical reactions (S, 2011). And their overload
from exogenous sources like, smoking, alcohol abuse,
UV radiations and air pollution added to the
endogenous production of free radicals that results in
oxidative stress and oxidative damage to the tissues
(Ghobashy et al., 2010; Mohod et al., 2014; Wu and
Cederbaum, 2003) as well as to DNA, proteins and
membrane lipids (Cosmas Achudume and Aina, 2012;
Wu and Cederbaum, 2003).
Under oxidative stress conditions, the antioxidant
enzyme levels are altered, in order to cope with the
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729 The role of Ficus virens Ait and its novel bioactive compound
tremendous increase in the production of ROS
(Anbarasi et al., 2006; McCord, 1993; Mohod et al.,
2014). However, after prolonged exposure, the toxic
effects of cigarette smoke emerge to override the
adaptive mechanism of the body tissues, as indicated
by a decrement in the levels of these enzymes (Hulea
et al., 1994). It is clear from the above discussion CS-
induces paramount oxidative stress and is consistent
with our results that demonstrate significant decrease
in liver and lung enzymatic activity of SOD, CAT,
Gred, GST including the GSH level, while Gpx activity
was significantly increased in CS-exposed rats.
In antioxidant defense system SOD is known to be the
first enzyme, responsible for scavenging the
superoxide radicals to form H2O2 (Andersen et al.,
1997). Furthermore, H2O2 is scavenged by
catalase/GSH (glutathione peroxidase) or it facilitate
in the development of highly reactive oxygen species
(Inoue et al., 2013; Mohod et al., 2014). The tar phase
of cigarette smoke contains quinone–semiquinone
radicals which are capable of reducing molecular
oxygen to superoxide radicals whose extreme
generation inactivates this enzyme (Church and Pryor,
1985; Durak et al., 2002). The proposed decrease in
SOD activity in CS-exposed rats could have resulted
from its inactivation by tar phase oxidants. The
presence and production of the free radicals from
smoke lower enzymatic activity of CAT, leading to
accumulation of H2O2 and lipid hydroperoxides which
further deteriorate the tissue damage (Pryor and
Stone, 1993). Inhibition of CAT activity in rat liver and
lung by cigarette smoke was observed during the
present study which is in conjunction of previous
reports (Luchese et al., 2009; Ramesh et al., 2007;
Ramesh et al., 2010).
Figure 5. Histology of lung section. A. Microphotograph of lung section from normal control rats, B. Microphotograph of lung
section from cigarette smoke-exposed rats after 4 weeks of cigarette smoke exposure, C. Microphotograph of lung section from
cigarette smoke-exposed rats after 4 weeks of FVBM-50 treatment, D. Microphotograph of lung section from cigarette smoke-
exposed rats after 4 weeks of FVBM-100 treatment, E. Microphotograph of lung section from cigarette smoke-exposed rats after 4
weeks of F18 bioactive compound treatment, F. Microphotograph of lung section from cigarette smoke-exposed rats after 4 weeks of
atorvastatin treatment. Figures were captured at 100X.
However, a decrease in the activity of CAT in the
present study suggests the in-vivo decrease in
antioxidant level which in turn unable to defend the
oxidative stress generated via cigarette smoke.
Treatment with FVBM extract and F18 bioactive
compound to smoke-exposed rats resulted in a
significant increase in both liver and lung SOD and
CAT activity which further strengthen the potent free
radical scavenging property of plant extract and
bioactive compound. Reduced glutathione play an
important role in glutathione-dependent antioxidant
system that most probably act as free radicals
scavenger or a substrate for Gpx and GST during the
detoxification of H2O2 (Masella et al., 2005).
Glutathione is maintained in body from its oxidized
form by the enzyme Gred, which requires NADPH as
Iqbal et al., 2016 Biomed Res Ther 2016, 3(7): 723-732
730 The role of Ficus virens Ait and its novel bioactive compound
a cofactor (Carmel-Harel and Storz, 2000). Previously
it has been reported that GSH was depleted during
CS-exposure in various tissues. Meanwhile, the
decrease in tissue GSH levels in CS-exposed rats may
be because of declined Gred activity and probably
reduced NADPH supply (Masella et al., 2005). GST is
involved in the detoxification of ROS and toxic
compounds from cigarette smoke, by conjugating
them to GSH. This conjugation reaction results in
depletion of the intracellular GSH that may further
enhances oxidant injury probably due to non-
availability of GSH for antioxidant enzymes such as
Gpx. Recent experimental data support the
assumption that CS exposure increases oxidative
stress and act as a potential mechanism for initiating
cardiovascular dysfunction (Ambrose and Barua,
2004).
Our data is well in agreement with above discussed
reports and observed the significant decline in GSH,
Gred and GST level in contrast the Gpx activity in
liver and lung of CS-exposed rats was significantly
increased which is due to inability of CAT to cope
with the oxidative stress. Helen and Vijayammal
(1997) also observed a similar decrease in the activity
of SOD, CAT, Gred and an increase in Gpx in rats
exposed to cigarette smoke. Gpx is also a scavenging
enzyme, but an increase in its activity in tissues of CS-
exposed rats may further reduce the GSH content. In
addition, an increased Gpx activity represents a
compensatory mechanism to degrade H2O2.
Simultaneous administration of FVBM extract (50 and
100 mg/rat) and F18 bioactive compound (1 mg/rat)
significantly increases CAT, SOD, Gred and GST
activity as well GSH concentration coupled with
decrease in Gpx level in CS-exposed stress rats. Thus,
it has been concluded that administration of FVBM
extract at higher dose and F18 bioactive compound to
CS-exposed rats restore the enzymatic (SOD, CAT,
Gred, GST and Gpx) and non-enzymatic (GSH)
antioxidant defense system and thus protect liver and
lung cells against oxidative stress-induced damage by
directly counteracting ROS/free radicals and by
activating the overall antioxidant defense systems.
Histological observations noticed the pulmonary con-
gestion and thickening of interalveolar septa in rats
exposed to cigarette smoke. The present study showed
devastation of some alveolar walls and foci of col-
lapsed alveoli with subsequent dilation of the adjoin-
ing alveolar spaces and development of large irregular
spaces (emphysematous changes). Cigarette smoking
causes endothelial dysfunction due to increased oxi-
dative stress (Messner and Bernhard, 2014). Our re-
sults coincided with those who interrelated the thick-
ening of the alveolar septa to modification in the vas-
cular bed resulting in inflammatory infiltration and
oedema (Hora et al., 2003). Chronic cigarette smoke
exposure causes lung tissue destruction might be due
to increased production of metalloproteinases (MMP)
proteolytic enzymes by macrophages (Barnes et al.,
2003; Taraseviciene-Stewart and Voelkel, 2008). The
liver showed congestion in central vein, portal in-
flammation and necrosis in CS-exposed rats. These
morphological changes were significantly reversed
after treatment with FVBM extract and F18 com-
pound. Thus, biochemical and histopathological stud-
ies suggested that, FVBM extract and F18 showed its
protective nature against CS-exposed rats.
CONCLUSION
In conclusion, our combined results clearly demon-
strated the protective role of FVBM extract and F18
compound in CS-induced severe oxidative stress. The
results are well supported by histopathological obser-
vations and indicates that F18/FVBM extract are
highly promising natural antioxidant as well as can be
used as an antioxidant and antiatherogenic agent.
However, further large-scale clinical trials in smokers
with and without coronary heart disease are required
to substantiate their antioxidative, atheroprotective
properties.
Acknowledgements
The Authors like to thank Prof. S.W. Akhtar, vice
chancellor, for providing state-of-the-art research
laboratory and animal house for smooth succession of
this work. The author would also like to thank
Deanship of Scientific Research at Majmaah
University for their support and contribution to this
study.
Iqbal et al., 2016 Biomed Res Ther 2016, 3(7): 723-732
731 The role of Ficus virens Ait and its novel bioactive compound
Competing Interests
The authors declare they have no competing interests.
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Cite this article as:
Iqbal, D., Khan, M., Khan, A., & Ahmad, S. (2016).
Extenuating the role of Ficus virens Ait and its novel
bioactive compound on antioxidant defense system
and oxidative damage in cigarette smoke exposed rats.
Biomedical Research and Therapy, 3(7), 723-732.